Although the specific role(s) for B cells in disease pathogenesis has not been established, B cells are strongly implicated in development of T1D both in animal models and in humans. Consistent with this idea, B cell depletion therapy with rituximab has recently been shown to be an effective treatment in some individuals with new onset T1D. Our published and preliminary studies indicate that healthy subjects, who carry the PTPN22 1858T variant allele strongly associated with T1D, exhibit impairment in B cell antigen receptor (BCR) signal transduction and altered B cell development. Strikingly, we have also identified nearly identical changes in B cell signaling and development in the majority of T1D subjects independent of their PTPN22 genotype. In addition, emerging data have also begun to implicate altered B regulatory (Breg) function in human autoimmune disorders. Together, these findings imply that altered B cell signaling comprises a common phenotype that participates in the pathogenesis of T1D. In this application, we propose to test the hypotheses that alterations in B cell signaling and phenotype: a) comprise a fixed deficit in at least a subset of T1D subjects that will be evident via natural history analysis of T1D cohorts;and b) may predict the response of T1D subjects to B cell depletion therapy with rituximab. We will test these hypotheses via two Specific Aims. First, we will characterize B cell signal transduction in individuals prior to and at the time of T1D disease onset. We will compare BCR-triggered p- PLCg2 and CD40L-driven of IL10 expression and pSTAT3, respectively, in blood samples derived from new onset T1D vs. age matched sibling controls using banked blood samples obtained from the TrialNet natural history study. We will determine if altered B cell signaling is present at disease onset as well as prior to clinical disease. Our data will also be characterized with respect to the overall composition of the B cell compartment and SNP genotyping for key autoimmune-associated gene products. Second, we will characterize B cell signaling in participants in the TrialNet rituximab study. We will compare B cell signaling and subsets prior to therapy in responders vs. non-responders to determine whether signaling correlates with response to therapy. Next, we will examine signaling activity in each subject using samples obtained at time 0 and 12 months post therapy. Further, we will address whether rituximab treatment modulates these defect(s) upon recovery of the B cell pool;and/or whether correction correlates with response to therapy. Finally, we will also characterize these outcomes with respect to SNP analysis. Linking our findings to TrialNet samples from both natural history and rituximab intervention studies provides a unique opportunity to test the idea that altered B cell signaling may promote a break in tolerance and/or modulate the response to tolerogenic therapies in T1D. If successful, this work will define new biomarkers for identifying and monitoring T1D patients that might benefit from future clinical trials using alternative targeting strategies to achieve long-term immune tolerance. PUBLIC HEALTH RELEVANCE: B cells are strongly implicated in development of T1D and B cell depletion therapy with rituximab has recently been shown to be an effective treatment in some individuals with new onset T1D. Our published and preliminary studies imply that altered B cell signaling comprises a common phenotype that participates in the pathogenesis of T1D. In this application, we will test the idea that alterations in B cell signaling: a) represent a fixed deficit in T1D subjects evident via natural history analyses;and b) may predict the response to B cell depletion therapy.